Abstract:

In this study, the use of the rpoB gene to identify mycobacterial species was evaluated. The rpoB gene encodes the highly conserved beta subunit of RNA polymerase. It is known to be more discriminatory in bacterial species identification compared to the widely used I6S rRNA gene sequence. This attribute can be exploited for correct identification of mycobacterial strains identified according to the 16S rRNA but requiring further identification to (sub-) species level. A gene fragment of approximately 760 bp was targeted using primer pair My9 - MycoR. The study involved evaluating the primer set on 30 reference mycobacterial strains from the public culture collection (BCCM/ITM). The tests were performed using an optimised in-house PCR system followed by gel electrophoresis and sequencing of the amplicons. BLAST analysis was performed to identify the species. Twenty-five (83%) of the strains were in agreement with the sequence data available from GenBank (NCBI). For three species namely Mycobacterium sphagni, M. komossense and M. parafortuitum, the sequence information was not available on GenBank (NCBI). Test attributes including repeatability and reproducibility were performed on two strains (M. ulcerans and M. abscessus) and results were consistent with tested strains. The rpoB was further evaluated on 71 mycobacterial strains from BCCM/ITM, which required further identification. According to the test results, thirty-one of the strains (43%) were identified to the respective species. Using the rpoB gene sequence, it was possible to distinguish M. marinum (1TM M000140/NCTC 2275) from M. ulcerans and M. montmartrense (NCBI strain 028/ITM 981197) from M. peregrinum and M. septicum which 16S rRNA is not able to do. The rpoB gene sequence has an added value in mycobacterial species identification. However, some rpoB gene sequences are not available on Genbank. It is therefore important to establish a complete rpoB gene sequence data bank for mycobacterial species especially the non-tuberculous mycobacteria (NTM) to improve their identification and to aid in disease diagnosis.